Manufacture of autogenous replacement body parts

ABSTRACT

Disclosed are matrix materials, methods, and devices for manufacture in vivo of autogenous replacement body parts comprising plural distinct tissues. In one embodiment, the replacement body part is a skeletal joint and the new plural distinct tissues include bone and articular cartilage.

RELATION TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/253,398,filed Jun. 3, 1994 now U.S. Pat. No. 5,906,827, the disclosure of whichis incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to materials and methods for the repair andregeneration of plural distinct tissues at a single defect site in amammal. More particularly, the invention is concerned with materials andmethods for the manufacture in vivo of autogenous replacement bodyparts, including mammalian skeletal joints, comprising plural differenttissues, such as ligament, articular cartilage and bone tissues.

BACKGROUND OF THE INVENTION

Skeletal joints provide a movable union of two or more bones. Synovialjoints are highly evolved articulating joints that permit free movementBecause mammalian lower limbs are concerned with locomotion and upperlimbs provide versatility of movement, most of the joints in theextremities are of the synovial type. There are various types ofsynovial joints. Their classification is based upon the types of activemotion that they permit (uniaxial, biaxial, and polyaxial). They aredifferentiated further according to their principal morphologicalfeatures (hinge, pivot, condyloid). In contrast to fibrous andcartilaginous joints where the ends of the bones are found in continuitywith intervening tissue, the ends of the bones in a synovial joint arein contact, but separate. Because the bones are not bound internally,the integrity of a synovial joint results from its ligaments and capsule(which bind the articulation externally) and to some extent from thesurrounding muscles. In synovial joints, the contiguous bony surfacesare covered with articular or, hyaline cartilage, and the joint cavityis surrounded by a fibrous capsule which segregates the joint from thesurrounding vascularized environment. The inner surface of the capsuleis lined by a synovial layer or "membrane" containing cells involved insecreting the viscous lubricating synovial fluid. Gray, Anatomy of theHuman Body, pp. 312; 333-336 (13th ed.; C. C. Clemente, ed., (1985)).

In certain synovial joints, the joint or synovial cavity may be dividedby a meniscus of fibrocartilage. Synovial joints involving two bones andcontaining a single joint cavity are referred to as simple joints.Joints that contain a meniscus forming two joint cavities are calledcomposite joints. The term compound joint is used for thosearticulations in which more than a single pair of articulating surfacesare present.

Joint replacement, particularly articulating joint replacement, is acommonly performed procedure in orthopedic surgery. However, the idealmaterial for replacement joints remains elusive. Typically, jointreconstruction requires repair of the bony defect, the articularcartilage and, in addition, one or more of the joining ligaments. Todate, there are no satisfactory clinical means for readily repairingboth articular cartilage and bony defects within a joint which reliablyresults in viable, fully-functional weight-bearing joints. Prostheticjoints which replace all the endogenous joint tissues circumvent some ofthese problems. However, prosthetic joints have numerous, welldocumented limitations, particularly in younger and highly activepatients. In addition, in some circumstances prosthetic jointreplacement is not possible and repair options are limited toosteochondroallograft materials.

The articular, or hyaline cartilage, found at the end of articulatingbones is a specialized, histologically distinct tissue and isresponsible for the distribution of load resistance to compressiveforces, and the smooth gliding that is part of joint function. Articularcartilage has little or no self-regenerative properties. Thus, if thearticular cartilage is torn or worn down in thickness or is otherwisedamaged as a function of time, disease or trauma, its ability to protectthe underlying bone surface is compromised.

Other types of cartilage in skeletal joints include fibrocartilage andelastic cartilage. Secondary cartilaginous joints are formed by discs offibrocartilage which join vertebrae in the vertebral column. Infibrocartilage, the mucopoly-saccharide network is interlaced withprominent collagen bundles and the chondrocytes are more widelyscattered than in hyaline cartilage. Elastic cartilage contains collagenfibers which are histologically similar to elastin fibers. As with otherconnective tissues the formation of cartilaginous tissue is a complexbiological process, involving the interaction of cells and collagenfibers in a unique biochemical milieu.

Cartilage tissue, including articular cartilage, unlike other connectivetissues, lacks blood vessels, nerves, lymphatics and basement membrane.Cartilage is composed of chondrocytes which synthesize an abundantextracellular milieu composed of water, collagens, proteoglycans andnoncollagenous proteins and lipids. Collagen serves to trapproteoglycans and to provide tensile strength to the tissue. Type IIcollagen is the predominant collagen in cartilage tissue. Theproteoglycans are composed of a variable number of glycosaminoglycanchains, keratin sulphate, chondroitin sulphate and/or dermatan sulphate,and N-linked and O-linked oligosaccharides covalently bound to a proteincore. The sulfated glycosaminoglycans are negatively charged resultingin an osmotic swelling pressure that draws in water.

In contrast, certain collagens such as the fibrotic cartilaginoustissues which occur in scar tissue for example, are keloid and typicalof scar-type tissue, i.e., composed of capillaries and abundant,irregular, disorganized bundles of Type I and Type II collagen.

Histologically, articular or hyaline cartilage can be distinguished fromother forms of cartilage, both by its morphology and by itsbiochemistry. Morphologically, articular cartilage is characterized bysuperficial versus mid versus deep "zones" which show a characteristicgradation of features from the surface of the tissue to the base of thetissue adjacent to the bone. In the superficial zone, for example,chondrocytes are flattened and lie parallel to the surface embedded inan extracellular network that contains tangentially arranged collagenand few proteoglycans. In the mid zone, chondrocytes are spherical andsurrounded by an extracellular network rich in proteoglycans andobliquely organized collagen fibers. In the deep zone, close to thebone, the collage fibers are vertically oriented. The keratin sulphaterich proteoglycans increase in concentration with increasing distancefrom the cartilage surface. For a detailed description of articularcartilage micro-structure, see, for example, (Aydelotte and Kuettner,(1988), Conn. Tiss. Res. 18: 205; Zanetti et al., (1985), J. Cell Biol.101: 53; and Poole et al., (1984), J. Anat. 138: 13.

Biochemically, articular collagen can be identified by the presence ofType II and Type IX collagen, as well as by the presence ofwell-characterized proteoglycans, and by the absence of Type X collagen,which is associated with endochondral bone formation.

In normal articular cartilage, a balance exists between synthesis anddestruction of the above-described extracellular network. However, intissue subjected to repeated trauma, for example due to friction betweenmisaligned bones in contact with one another, or in joint diseasescharacterized by net loss of articular cartilage, e.g., osteoarthritis,an imbalance occurs between synthesis and degradation.

Two types of defects are recognized in articular surfaces, i.e.,full-thickness defects and superficial defects. These defects differ notonly in the extent of physical damage to the cartilage, but also in thenature of the repair response each type of lesion can elicit.

Full-thickness defects of an articulating surface include damage to thehyaline cartilage, the calcified cartilage layer and the subchondralbone tissue with its blood vessels and bone marrow. Full-thicknessdefects can cause severe pain since the bone plate contains sensorynerve endings. Such defects generally arise from severe trauma and/orduring the late stages of degenerative joint disease, such asosteoarthritis. Full-thickness defects may, on occasion, lead tobleeding and the induction of a repair reaction from the subchondralbone. In such instances, however, the repair tissue formed is avascularized fibrous type of cartilage with insufficient biomechanicalproperties, and does not persist on a long-term basis.

In contrast, superficial defects in the articular cartilage tissue arerestricted to the cartilage tissue itself. Such defects are notoriousbecause they do not heal and show no propensity for repair reactions.Superficial defects may appear as fissures, divots, or clefts in thesurface of the cartilage, or they may have a "crab-meat" appearance inthe affected tissue. They contain no bleeding vessels (blood spots) suchas are seen in full-thickness defects. Superficial defects may have noknown cause, however, they are often the result of mechanicalderangements which lead to a wearing down of the cartilaginous tissue.Such mechanical derangements may be caused by trauma to the joint, e.g.,a displacement of torn meniscus tissue into the joint, meniscectomy, alaxation of the joint by a torn ligament, malalignnment of joints, orbone fracture, or by hereditary diseases. Superficial defects are alsocharacteristic of early stages of degenerative joint diseases, such asosteoarthritis. Since the cartilage tissue is not innervated orvascularized, superficial defects do not heal and often degenerate intofull-thickness defects.

Replacement with prosthetic joints is currently the preferred option forserious degeneration of joint function involving loss of articularcartilage. It is anticipated that a means for functional reconstructionof joint complexes, including regeneration and repair of articularcartilage, will have a profound effect on alloplastic joint replacementsurgery and the management of degenerative joint disease.

Like articular cartilage, joint ligaments which serve to connectinteracting bones in the joint, have little or no self-regenerativeproperties. Ligaments typically are composed of substantially parallelbundles of white fibrous tissue. They are pliant and flexible to allowsubstantially complete freedom of movement, but are inextensile toprevent over-extension of the interacting bones in the joint. Likecartilage, ligament tissue is substantially devoid of blood vessels andhas little or no self-regenerative properties. Surgical repair of tornor damaged ligament tissue to date is limited to use of autogenousgrafts or synthetic materials that are surgically attached to thearticular extremities of the bones. Allogenic ligaments typically failmechanically, presumably due to the treatments required to render thesematerials biocompatible. Similarly, tendons are rope-like structureswhich connect muscle fibers to bone or cartilage and which are formedfrom substantially parallel fibroids of white connective tissue. Thesynovial capsule is composed of a thin layer of ligamentous tissue whichencloses the joint and allows the joint to be bathed in the lubricatingsynovial fluid. The interior of the joint capsule is lined with a thinmembrane of connective tissue having branched connective-tissuecorpuscles defining the synovial membrane, and which is primarilyresponsible for secreting synovial fluid into the cavity. The integrityof this membrane therefore, is important to maintaining a source for thelubricating synovial fluid. Repair of these tissues in orthopediccontexts typically is limited to resuturing of existing tissue.

Bone tissue differs significantly from the other tissues describedhereinabove, including cartilage tissue. Specifically, bone tissue isvascularized tissue composed both of cells and a biphasic medium whichis composed of a mineralized, inorganic component (primarilyhydroxyapatite crystals) and an organic component comprised primarily ofType I collagen. Glycosaminoglycans constitute less than 2% of thisorganic component and less than 1% of the biphasic medium itself or ofbone tissue per se. Moreover, relative to cartilage tissue, the collagenpresent in bone tissue exists in a highly-organized parallelarrangement.

Bony defects, whether from degenerative, traumatic or cancerousetiologies, pose a formidable challenge to the reconstructive surgeon.Particularly difficult is reconstruction or repair of skeletal partsthat comprise part of a multi-tissue complex, such as occurs inmammalian joints.

Mammalian bone tissue is known to contain one or more proteinaceousmaterials presumably active during growth and natural bone healing whichcan induce a developmental cascade of cellular events resulting inendochondral bone formation. The developmental cascade involved inendochondral bone differentiation consists of chemotaxis of mesenchymalcells, proliferation of progenitor cells into chondrocytes andosteoblasts, differentiation of cartilage, vascular invasion, boneformation, remodeling, and finally marrow differentiation.

True osteogenic factors capable of inducing the above-described cascadeof events that result in endochondral bone formation have now beenidentified, isolated, and cloned. These proteins, which occur in natureas disulfide-bonded dimeric proteins, are referred to in the art as"osteogenic" proteins, "osteoinductive" proteins, and "bonemorphogenetic" proteins. Whether naturally-occurring or syntheticallyprepared, these osteogenic proteins, when implanted in a mammaltypically in association with a substrate that allows the attachment,proliferation and differentiation of migratory progenitor cells, arecapable of inducing recruitment of accessible progenitor cells andstimulating their proliferation, inducing differentiation intochondrocytes and osteoblasts, and further inducing differentiation ofintermediate cartilage, vascularization, bone formation, remodeling, andfinally marrow differentiation. Those proteins are referred to asmembers of the Vgr-1/OP1 protein subfamily of the TGFβ super gene familyof structurally related proteins. Members include the proteins describedin the art as OP1 (BMP-7), OP2 (BMP-8), BMP2, BMP3, BMP4, BMP5, BMP6,60A, DPP, Vgr-1 and Vg1. See., e.g., U.S. Pat. No. 5,011,691; U.S. Pat.No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093, Wharton etal. (1991) PNAS 88: 9214-9218), (Ozkaynak (1992) J. Biol. Chem. 267:25220-25227 and U.S. Pat. No. 5,266,683); (Celeste et al. (1991) PNAS87: 9843-9847); (Lyons et al. (1989) PNAS 86: 4554-4558). Thesedisclosures describe the amino acid and DNA sequences, as well as thechemical and physical characteristics of these proteins. See also(Wozney et al. (1988) Science 242: 1528-1534); BMP 9 (WO93/00432,published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325: 81-84;and Vg-1 (Weeks (1987) Cell 51: 861-867).

It is an object of the instant invention to provide a bioresorbablematrix and device, suitable for regenerating body parts which comprisetwo or more functionally- and structurally-associated yet distinctreplacement tissues in a mammal. Another object is to providecompositions and methods for the repair or complete reconstruction of amechanically and functionally viable skeletal joint in a mammal,particularly an articulating or synovial joint, as well as other bodyparts comprising bone and bona fide hyaline cartilage, without relyingon prosthetic devices. Another object is to provide materials andmethods for the repair of tissue defects in an articulating mammalianjoint, so as to form a mechanically and functionally viable jointcomprising bone and articular cartilage, ligament, tendon, synovialmembrane and synovial capsule tissue. Another object of the invention isto provide means for restoring functional non-mineralized tissue in askeletal joint including the avascular tissue therein.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods and devices areprovided for the manufacture of a live autogenous replacement partcomprising plural distinct tissues. In one aspect the replacement bodypart includes part or all of a mammalian skeletal joint, including anarticulating or synovial joint. As described herein below, the methodsand compositions of the invention are sufficient to restore mechanicaland functional viable of the tissues associated with a skeletal joint,including bone (and bone marrow), articular cartilage, ligament, tendon,synovial capsule and synovial membrane tissues. Thus the inventionprovides methods and compositions for replacement of one or more of theplural distinct tissues that define a mammalian skeletal joint.

The invention provides, in one aspect therefore, a novel matrix forforming a mechanically and structurally functional, mammalian,replacement body part comprising plural distinct tissues. The matrixcomprises intact residues specific for or characteristic of, and/orderived from at least two distinct tissues of the replacement body part.As will be appreciated from the description provided herein below, thematrix can include residues specific for four or more distinct tissues.The matrix is biocompatible and bioresorbable. Specifically, it issufficiently free of pathogens and antigenic stimuli that can result ingraft rejection. Preferably the matrix is derived from an allogenic orxenogenic body part. Preferably, it is derived from a mammalian donor,such as a cadaver. The body part may be rendered inert or "devitalized"by dehydration, such as by ethanol extraction and lyophilization, sothat no residual cellular metabolism remains, but the function ofendogenous growth factors and the like can be restored upon in situreconstitution by endogenous body fluids. The treated body part whichnow is substantially depleted in antigenic and pathogenic components andnow is biocompatible, maintains the residues specific for the pluraldistinct tissues constituting the body part sought to be replaced. Theseresidues include those of plural distinct tissues with dimensions andstructural relationships to each other which mimic those of the bodypart to be replaced.

The thus treated matrix having utility in the methods and devices of theinvention lacks significant mechanical integrity as compared with nativetissue and, on its own, is not sufficient to induce regeneration of areplacement body part or tissue when implanted. However, by impregnatingor otherwise infusing the interstices of the matrix with osteogenicprotein so that the protein is disposed on or adsorbed to, the surfacesof the matrix, the device of the instant invention is formed and issufficient to induce formation of new tissue in vivo such thatregeneration of a mechanically and functionally viable replacement bodypart occurs in situ.

In one preferred embodiment, the device comprises part or all of askeletal joint excised from a mammalian donor allogenic or xenogenic tothe donee. Treated as described herein the device comprising theallogenic or xenogenic skeletal joint (1) is biocompatible, namely, itis non-pathogenic and sufficiently non-antigenic to prevent graftrejection in vivo, and (2) is sufficient to induce formation of afunctionally viable autogenous replacement joint in vivo, includinggenerating functional bone, articular cartilage, ligament and capsuletissue in correct relation to one another such that a structurally andmechanically functional replacement joint results.

In another embodiment, the invention provides a device which serves as atemplate for forming in vivo part or all of a skeletal synovial jointcomprising plural distinct tissues and which, in response to morphogenicsignals, induces new tissue formation, including new articular cartilagetissue from responding cells present in the synovial environment. Thenewly formed tissues assume the shape and function of the originaltissue in the skeletal joint.

In another aspect, the invention provides methods for replacing adefective body part comprising the steps of: excising the defective bodypart and implanting the device of the instant invention. In oneembodiment, the method also comprises the additional step of providing asupply of mesenchymal cells to the implanted device, as by threading orotherwise providing a muscle flap prefused with a blood supply into ahollow portion of the device. In another embodiment, the device isimplanted at a locus in the body of the individual distinct from thedefect site but which allows generation of the replacement body part.The autogenous body part thus formed then can be implanted at the defectsite.

As will be appreciated from the description provided herein, in anotheraspect, the invention provides devices and methods for the functionaland mechanical restoration of one or more individual tissues in amammalian skeletal joint, including the non-mineralized and avasculartissue therein. Thus, in one embodiment, the invention provides methodsand devices competent for restoring, without limitation, functionalarticular cartilage, ligament, synovial membrane and synovial capsuletissue. The methods and devices described herein can be used forexample, to correct superfical articular cartilage defects in a joint,to replace torn or compromised ligaments and/or tendons, and to repairdefects in synovial capsule or membrane tissue.

The devices for repairing individual skeletal joint tissue compriseosteogenic protein disposed on a matrix containing residues specificfor, or derived from skeletal joint tissue of the type to be restored,including, without limitation, cartilage, ligament, tendon, synovialcapsule, or synovial membrane tissue. The device can take the form of asolid, or it can have the physical properties of a paste or gel.Preferably, the matrix is derived from allogenic or xenogenic tissue,and is treated as described herein to form a biocompatible devitalizedmatrix.

In another embodiment the matrix can be formulated de novo fromsynthetic and/or naturally-derived components. The matrix includes both(a) residues specific for, or characteristic of, the given tissue and,(b) materials sufficient to create a temporary scaffold for infiltratingcells and defining a three dimensional structure which mimics thedimensions of the desired replacement tissue. Useful such materials aredescribed herein below. Suitable tissue-specific residues can beobtained from devitalized allogenic or xenogenic tissue and combinedwith the structural materials as described herein to create thesynthetic matrix. In another embodiment, the matrix comprisesdevitalized non-mineralized tissue. In some circumstances, as in theformation of articular cartilage on subchondral bone, a non-mineralizedmatrix material defining a three-dimensional structure which allows theattachment of infiltrating cells, can be sufficient, in combination withosteogenic protein, to induce new tissue formation

While, as described above, in a preferred embodiment the inventioncontemplates a device suitable as a template for forming in vivo areplacement skeletal joint, as will be appreciated by the practioner inthe art, the invention contemplates, and the disclosure enables, adevice suitable as a template for forming in vivo functional replacementbody parts other than skeletal joints and which comprise plural distincttissues.

When used in accordance with the methods of the instant invention, thedevices of the invention and/or the tissues which result from theirapplication, essentially satisfy the following criteria of a preferredgrafting material:

1. They result in formation of mechanically and functionally viabletissues normally present at the site. These tissues are of anappropriate size and have correct structural relationships so as toresult in a functional body part. In particular, the multi-tissuereplacement part, whether produced in situ at the site of intended useor remotely, becomes incorporated, integrating with adjacent tissues,essentially maintaining its shape, and avoiding abnormal resorption,regardless of the conditions present at the recipient site. Weiland etal. (1983) Clin. Orthop. 174: 87 (1983).

2. The devices are capable of being precisely contoured and shaped toexactly match any defect, whichever complex skeletal or organ shape itis meant to replace.

3. The devices virtually have unlimited supply and are relatively easyto obtain.

4. The devices have minimal donor site morbidity.

Furthermore, the instant invention provides practitioners with materialsand methods for skeletal joint repair including the repair of the boneand articular cartilage present therein, and which solve problems thatoccur using the methods and devices of the art. For example, the instantinvention can induce formation of bona fide hyaline cartilage ratherthan fibrocartilage at a defect site. Using the materials and methodsdisclosed herein, functional hyaline cartilage forms on the articulatingsurface of bone at a defect site and does not degenerate over time tofibro-cartilage. By contrast, prior art methods of repairing cartilagedefects generally ultimately result in development of fibrous cartilageat the defect site. Unlike hyaline cartilage, fibrocartilage lacks thephysiological ability to restore articulating joints to their fullcapacity. Thus, when the instant materials are used in accordance withthe instant methods, the practitioner can substantially functionallyrestore a cartilage defect in an articulating joint, particularly asuperficial articular cartilage defect and substantially avoid theundesirable formation of fibrocartilage typical of prior art methods, ordegeneration into a "full-thickness defect". The invention also providesmeans for repairing individual tissue of a joint not readily reparableindividually using prior art methods, and which, in some cases,previously warranted replacement of the entire joint with a prostheticdevice. The invention further allows use of allogenic replacementmaterials for repairing the avascular tissue in a skeletal joint, andwhich result in the formation of mechanically and functionally viablereplacement tissues at a joint locus.

BRIEF DESCRIPTION OF THE DRAWINGS

While the specification concludes with claims particularly pointing outand specifically claiming the subject matter which is regarded asconstituting the present invention, it is believed that the inventionwill be better understood from the following detailed description ofpreferred embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a fragmentary front elevational view of a mammalian knee jointwith sufficient tissue removed to show the articular cartilage on thecondyles of the femur, the ligaments, synovial membrane, joint capsule,and further showing a damaged area in the articular cartilage requiringrepair;

FIGS. 2A through 2D are schematic representations of the elements usedto generate a viable, functional glenohumoral hemi-joint in oneembodiment of the invention. FIG. 2A depicts a lyophilized allograft;FIG. 2B depicts osteogenic protein for application to the lyophilizedallograft of FIG. 2A; FIG. 2C depicts a muscle flap of cutaneous maximusmuscle to be threaded inside the shaft of the lyophilized allograft;and, FIG. 2D depicts a viable, functional hemi-joint resulting from thecombination of elements in FIGS. 2A, 2B and 2C. FIG. 2D represents oneembodiment of the device of the instant invention;

FIGS. 3A through 3D are schematic representations of the four allograftstested in the hemi-joint of Example 2 (5 week); and

FIGS. 4A through 4D are schematic representations of the four allograftstested in the hemi-joint of Example 3 (6 month).

DETAILED DESCRIPTION

In accordance with the present invention, novel materials and methodsare provided for the repair and regeneration of plural distinct tissues,including manufacture of a live autogenous replacement part comprisingplural distinct tissues. In one embodiment the replacement body part isa skeletal joint, particularly an articulating joint, and includes,without limitation, residues specific for, or derived from, bone,cartilage, ligament, tendon, synovial capsule and synovial membranetissue.

More particularly, in one aspect, the invention provides a devicecomprising an osteogenic protein disposed on the surfaces of a matrix orsubstrate for forming a functional, mammalian replacement body partcomprising plural distinct tissues. As used herein, the term "matrix" isunderstood to define a structure having interstices for the attachment,proliferation and differentiation of infiltrating cells. It comprisesresidues specific for the tissue to be replaced and/or derived from thesame tissue type, and has a shape and dimension when implanted whichsubstantially mimics that of the replacement tissue desired.

As used herein, the term "residue" is intended to mean a constituent ofa given tissue, which has specificity for, or is characteristic of, thegiven tissue, and which is derivable from the non-viable constituents ofthe given tissue. A matrix comprising these residue(s), when combinedwith osteogenic protein, and implanted in a mammal in an environmentwhich mimics the tissue's local environment under physiologicalconditions, and is sufficient for formation of specific, mechanicallyand functionally viable replacement tissue.

The term "plural distinct tissue" is intended to mean physiologicallydistinguishable tissues, such as biochemically or ultrastructurallydistinguishable tissues which reside at an anatomically similar locus.In an articulating replacement joint device for example, the matrix cancomprise residues specific for, or derived from, bone, cartilage,ligament, tendon and synovial membrane tissue. Thus, a significantaspect of the matrix of the invention is a single structure comprisingresidues of plural, distinct tissues, and which, when combined with anosteogenic protein as defined herein, is suitable for inducing repair orregeneration of a body part that is mechanically and functionally viableover time in vivo.

As used herein, the terms "bone" and "articular cartilage" are intendedto mean the following: Bone refers to a calcified (mineralized)connective tissue primarily comprising a composite of deposited calciumand phosphate in the form of hydroxyapatite, collagen (predominantlyType I collagen) and bone cells, such as osteoblasts, osteocytes andosteoclasts, as well as to the bone marrow tissue which forms in theinterior of true endochondral bone. Cartilage refers to a type ofconnective tissue that contains chondrocytes embedded in anextracellular network comprising fibrils of collagen (predominantly TypeII collagen along with other minor types, e.g. Types IX and XI), variousproteoglycans (e.g., chondroitin sulfate, keratan sulfate, and dermatansulfate proteoglycans), other proteins, and water. Articular cartilagerefers to hyaline or articular cartilage, an avascular, non-mineralizedtissue which covers the articulating surfaces of the portions of bonesin joints and allows movement in joints without direct bone-to-bonecontact, and thereby prevents wearing down and damage to opposing bonesurfaces. Most normal healthy articular cartilage is referred to as"hyaline," i.e., having a characteristic frosted glass appearance. Underphysiological conditions, articular cartilage tissue rests on theunderlying, mineralized bone surface, the subchondral bone, whichcontains highly vascularized ossicles. These highly vascularizedossicles can provide diffusible nutrients to the overlying cartilage,but not mesenchymal stem cells.

"Ligament" is intended to mean both the rope-like structures of whitefibrous connective tissue which attach anterior extremities ofintracting bones, as well as the tissue defining a synovial capsule."Synovial membrane" is intended to define the connective tissue membranelining the interior of the synovial cavity and which is involved insynovial fluid secretion. "Tendon" is intended to define the connectivetissue structure which joins muscle to bone.

Replacement Body Parts

As disclosed herein, the instant invention provide methods andcompositions for replacing and repairing a defective body part. Themethod comprises the steps of surgically excising the defective bodypart, implanting a device comprising a matrix of the type describedabove at the site of excision, and, as necessary, surgically repairingtissues adjacent the site of excision as described herein below. Forexample, for synovial joint replacement, it is desirable to repair thejoint capsule, including the synovial membrane and ligaments, so as tosurgically approximate the joint structure as it occurs physiologicalconditions, thereby recreating the avascular environment which is thesynovial cavity and which is bathed in synovial fluid. It also ispreferable to suture or otherwise mechanically temporarily connect theimplanted device to surrounding tissue.

In one embodiment the device is constructed to replace part or all of amammalian skeletal joint structure and includes a matrix having residuesfor plural, distinct tissues, including two or more of bone, cartilage,ligament, tendon, synovial capsule and/or synovial membrane tissue.

In another embodiment the device is constructed to replace an individualtissue of a mammalian skeletal joint, including an individual avascularand/or non-mineralized tissue. As demonstrated herein, the device iscompetent to induce functional replacement tissue formation, includingarticular cartilage, from responding cells present in the localenvironment, including a synovial environment, and without requiringcellular infiltration of mesenchymal cells from a vascularized muscleflap. The matrix of this embodiment comprises residues specific for, orcharacteristic of, and/or derived from, tissue of the same type as theindividual tissue to be replaced. In another embodiment, the matrixcomprises devitalized non-mineralized tissue. In a preferred embodiment,the replacement tissue can include articular cartilage, ligament, bone,tendon or synovial capsule tissue.

In a partial or complete joint replacement, it is preferred but notrequired to include in the practice of the method the additional step ofthreading a muscle flap into a hollow portion of the implanted device.For example, using the method described in Khouri, U.S. Pat. No.5,067,963, the disclosure of which is incorporated herein by referenceand herewith below, a muscle flap, which can itself be pretreated withosteogenic protein, can be surgically introduced into a cavity in theimplanted matrix, such as the marrow cavity of devitalized bone, toprovide a blood supply to expedite morphogenesis of vascularized tissueand to provide a ready supply of mesenchymal stem cells.

The matrix of instant invention has utility as an implantable devicewhen osteogenic protein is disposed on the surfaces of the matrix,present in an amount sufficient to induce formation of each of thereplacement tissues. This permits regeneration of the body part withinthe mammal, including plural tissues of appropriate size,interrelationship, and function. Osteogenic proteins contemplated to beuseful in the instant invention are described below and have beenearlier-described in, for example, U.S. Pat. Nos. 4,968,550, 5,258,499and 5,266,683, the disclosures of which are incorporated by referenceherein. The osteogenic protein can be, for example, any of the knownbone morphogenetic proteins and/or equivalents thereof described hereinand/or in the art and includes naturally sourced material, recombinantmaterial, and any material otherwise produced which is capable ofinducing tissue morphogenesis.

The methods and materials of the instant invention are especially usefulfor the repair and/or partial or complete replacement of mammalian bodyjoints, including, without limitation, articulating joints, particularlyjoints enclosed by a ligamentous capsule and bathed in synovial fluid.

In some synovial joints, the movement is uniaxial, i.e., all movementstake place around one axis: Among these are the ginglymus or hinge jointin which the axis of movement is transverse to the axes of the bones,and the trochoid or pivot joint in which the axis is longitudinal. Inthe case of biaxial synovial joints, movements are around two axes at aright angle or any other angle to each other: These include thecondyloid, the ellipsoid, and the saddle joints. There is a third typeof synovial joint, the spheroidal or ball-and-socket joint, in which themovements are polyaxial, i.e., movements are permitted in an infinitenumber of axes. Finally, there are the plane or gliding-type synovialjoints.

In hinge joints, the articular surfaces are molded to each other in sucha manner as to permit motion in only one plane around the transverseaxis. Flexion at the elbow joint is an example; other examples includethe interphalangeal joints of both the fingers and toes. In pivotjoints, movement in a pivot joint also occurs around a single axis,however, it is the longitudinal axis. There are several pivot joints inthe human body, such as the proximal radioulnar articulation. Incondylar joints include, movement occurs principally in one plane. Thetibiofemoral articulation of the knee joint is an example. In ellipsoidjoint include, movement is around two principal axes which are at rightangles to each other. Examples of these joints include the radiocarpaland metacarpophalangeal joints. In a saddle joint, the articular end ofthe proximal bone is concave in one axis and convex in a perpendicularaxis. These surfaces fit reciprocally into convex and concave surfacesof the distal bone. The best example of a saddle joint is thecarpometacarpal joint of the thumb. A ball-and-socket joint is one inwhich the distal bone is capable of motion around an indefinite numberof axes with one common center. Examples of this form of articulationare found in the hip and shoulder joints. A plane or gliding-type jointallows a slight slipping or sliding of one bone over the other. Unlikethe above-described joints, the amount of motion between the surfaces islimited by the ligaments or osseous processes that surround thearticulation. This is the form present in the joints between thearticular processes of certain vertebrae, the carpal joints, and theintermetatarsal joints.

Although it is contemplated that the present invention is usable torepair defects including bone and articular cartilage elsewhere in amammalian body, aspects of the invention are here illustrated inconnection with the articulating surfaces on the femur in a knee joint10 illustrated in FIG. 1.

FIG. 1 illustrates a knee joint 10 between the bottom of a femur 11 andthe top of a tibia 12. For clarity of illustration, only portions 13 and14 of the medial and lateral collateral ligaments which movably tie thefemur 11 to the underlying tibia 12 and fibula 15, are shown in FIG. 1.Similarly, the joint capsule is represented by the exterior dark lining25, and the synovial membrane, which lines the synovial cavity andsecretes the lubricating synovial fluid, is represented by the interiordark lining 26. Normally interposed between the opposing surfaces of thefemur 11 and tibia 12 are lateral and medial meniscus cartilages 16 and17 and anterior and posterior cruciate ligaments (not shown). Theconvexly curved condyles 20 and 21 at the lower end of the femur 11 arenormally supported by the meniscus cartilages 16 and 17, respectively,on the upper end of the tibia 12. Normally, the lower end of the femur11, including the condyles 20 and 21, are covered by a layer 22 ofhyaline cartilage material, referred to as the articular cartilage 22.The articular cartilage 22 forms a generally resilient padding which isfixed on the surface of the lower end of the femur 11 to protect thelatter from wear and mechanical shock. Moreover, the articular cartilage22, when lubricated by the synovial fluid in the knee joint 10, providesa surface which is readily slidable on the underlying surfaces of themeniscus cartilages 16 and 17 (or on the upper surface of the tibia 12should one or both of the meniscus cartilage 16 and 17 be partly ortotally absent) during articulation of the knee joint 10.

A portion of the articular cartilage may become damaged by injury ordisease, or become excessively worn. FIG. 1 illustrates an example of adamaged area 23.

Matrix Considerations

As will be appreciated by the skilled artisan, provided the matrix has athree dimensional structure sufficient to act as a scaffold forinfiltrating cells, and includes the residues specific for, orcharacteristic of, and/or which are derived from, the same tissue typeas the tissue to be repaired, the precise nature of the substrate per seused for the matrices disclosed herein is not determinative of amatrix's ultimate ability to repair and regenerate replacement tissue.In the instant invention, the substrate serves as a scaffold upon whichcertain cellular events, mediated by an osteogenic protein, necessarilywill occur. The specific responses to the osteogenic protein ultimatelyare dictated by the endogenous microenvironment at the implant site andthe developmental potential of the responding cells. As also will beappreciated by the skilled artisan, the precise choice of substrateutilized for the matrices disclosed herein will depend, in part, uponthe type of defect to be repaired, anatomical considerations such as theextent of vascularization at the defect site, and the like.

The matrix of the invention may be obtained as follows. A replacementtissue or body part to be used as a replacement body part and whichcomprises at least two distinct tissues in association to form the bodypart, is provided, as from a cadaver, or from a bone bank and treated,as by ethanol treatment and dehydrated by lyophilization, so that theremaining material is non-pathogenic and sufficient non-antigenic toprevent graft rejection. As described above, the thus treated materialhaving utility in the devices of the invention further comprises theresidues of the extracted tissue or tissues from which it is derived. Areplacement body part matrix thus treated further is dimensioned suchthat the residues have a structural relationship to each other whichmimic that of the body part to be replaced.

Natural-sourced Matrices

Suitable allogenic or xenogenic matrices can be created as describedherein below, using methods well known in the art. Preferably, thereplacement body part or tissue is obtained fresh, from a cadaver orfrom a tissue bank which freezes its tissues upon harvest. In all casesand as will be appreciated by the practitioner in the field, it ispreferable to freeze any tissue upon harvest, unless the tissue is to beput to immediate use. Prior to use, the tissue is treated with asuitable agent to extract the cellular non-structural components of thetissue so as to devitalize the tissue. The agent also should be capableof extracting any growth inhibiting components associated with thetissue, as well as to extract or otherwise destroy any pathogens. Theresulting material is an acellular matrix defining interstices that canbe infiltrated by cells, and is substantially depleted innon-structurally-associated components.

In a currently preferred procedure, the tissue is devitalized followinga methodology such as that used in the art for fixing tissue. The tissueis exposed to a non-polar solvent, such as 100% (200 proof) ethanol, fora time sufficient to substantially replace the water content of thetissue with ethanol and to destroy the cellular structure of the tissue.Typically, the tissue is exposed to 200 proof ethanol for several days,at a temperature in the range of about 4°-40° C., taking care to replacethe solution with fresh ethanol every 6-12 hours, until such time as theliquid content of the tissue comprises 70-90% ethanol. Typically,treatment for 3-4 days is appropriate. The volume of liquid added shouldbe more than enough to submerge the tissue. The treated tissue then islyophlized. The resulting, dry matrix is substantially depleted innon-structural components but retains both intracellular andextracellular matrix components derived from the tissue.

Numerous other methods are described in the art for extracting tissues,including mineralized tissue such as bone, and for rendering thesetissues biocompatible for allogenic or xenogenic implants. See, forexample, Sampath et al. (1983) PNAS 80: 6591-6595, U.S. Pat. No.5,011,691, and U.S. Pat. Nos. 4,975,526 and 5,171,574. Thesepublications describe extraction with 4M guanidine-HCl, 50 mM Tris-HCl,pH 7.0 for 16 hours at 4° C., and various deglycosylating and collagenfibril modifying agents, including hydrogen fluoride, trifluoroceticacid, dichloromethane, acetonitrile, isopropanol, heated, acidic aqueoussolutions, and various combinations of these reagents. The disclosuresof the patents is incorporated herein by reference. As described thereinand below, where the matrix is treated with a fibril-modifying agent,the treated matrix can be washed to remove any extracted components,following a form of the procedure set forth below:

1. Suspend matrix preparation in TBS (Tris-buffered saline) 1 g/200 mland stir at 4° C. for 2 hrs; or in 6 M urea, 50 mM Tris-HCl, 500 mMNaCl, pH 7.0 (UTBS) or water and stir at room temperature (RT) for 30minutes (sufficient time to neutralize the pH);

2. Centrifuge and repeat wash step; and

3. Centrifuge; discard supernatant; water wash residue; and thenlyophilize.

Treated allogenic or xenogenic matrices are envisioned to haveparticular utility for creating devices for forming replacement bodyparts comprising plural distinct tissues, as well as for creatingdevices for replacing individual joint tissues, such as ligament andarticular cartilage tissue. For example, a replacement ligament devicecan be formulated from an allogenic ligament matrix and osteogenicprotein, and implanted at a skeletal joint locus following standardsurgical procedures for autogenous ligament replacement. Similarly, anallogenic articular cartilage device can be formed from devitalizedcartilage tissue, or other inert, non-mineralized matrix material andosteogenic protein, and the device laid on the subchondral bone surfaceas a sheet. Alternatively, a formulated device can be pulverized orotherwise mechanically abraded to produce particles which can beformulated into a paste or gel as described herein for application tothe bone surface.

Synthetic Matrices

As an alternative to a natural-sourced matrix, or as a supplement to beused in combination with a natural-sourced matrix, a suitable matrixalso can be formulated de novo, using (1) residues derived from and/orcharacteristic of, or specific for, the same tissue type as the tissueto be repaired, and (2) one or more materials which serve to create athree-dimensional scaffolding structure that can be formed or molded totake on the dimensions of the replacement tissue desired. In somecircumstances, as in the formation of articular cartilage on asubchondral bone surface, osteogenic protein in combination with amatrix defining a three-dimensional scaffolding structure sufficient toallow the attachment of infiltrating cells and composed of anon-mineralized material can be sufficient. Any one or combination ofmaterials can be used to advantage, including, without limitation,collagen; homopolymers or copolymers of glycolic acid, lactic acid, andbutyric acid, including derivatives thereof; and ceramics, such ashydroxyapatite, tricalcium phosphate and other calcium phosphates andcombinations thereof.

The tissue-specific component of a synthetic matrix readily can beobtained by devitalizing an allogenic or xenogenic tissue as describedabove and then pulerizing or otherwise mechanically breaking down theinsoluble matrix remaining. This particulate material then can becombined with one or more structural materials, including thosedescribed herein. Alternatively, tissue-specific components can befurther purified from the treated matrix using standard extractionprocedures well characterized in the art and, using standard analysisprocedures, the extracted material at each purification step can betested for its tissue-specificity capability. See, for example, Sampathet al. (1987) PNAS 78: 7599-7603 and U.S. Pat. No. 4,968,590 forexemplary tissue extraction protocols.

A synthetic matrix may be desired where, for example, replacementarticular cartilage is desired in an existing joint to, for example,correct a tear or limited superficial defect in the tissue, or toincrease the height of the articular cartilage surface now worn due toage, disease or trauma. Such "resurfacing" of the articular cartilagelayer can be achieved using the methods and compositions of theinvention by, in one embodiment, treating a sheet of allogenic orxenogenic articular cartilage tissue as described herein, coating theresulting matrix with osteogenic protein, rolling up the formulateddevice so that it can be introduced to the joint using standardorthoscopic surgical techniques and, once provided to the site,unrolling the device as a layer onto the articular bone surface. Inanother embodiment, the device is formulated as a paste or injectablegel-like substance that can be injected onto the articular bone surfacein the joint also using standard orthoscopic surgical techniques. Inthis embodiment, the formulation may comprise a pulverized or otherwisemechanically degraded device comprising both matrix and osteogenicprotein and, in addition, one or more components which serve to bind theparticles into a paste-like or gel-like substance. Binding materialswell characterized in the art include, for example,carboxymethylcellulose, glycerol, polyethyleneglycol and the like.Alternatively, the device can comprise osteogenic protein dispersed in asynthetic matrix which provides the desired physical properties. As anexample, a synthetic matrix having tissue specificity for cartilage andbone is described in WO91/18558, published Dec. 21, 1991 and hereinbelow. Briefly, the matrix comprises a porous crosslinked structuralpolymer of biocompatible, biodegradable collagen and appropriate,tissue-specific glycosaminoglycans as tissue-specific cell attachmentfactors. Collagen derived from a number of sources can be used,including insoluble collagen, acid-soluble collagen, collagen soluble inneutral or basic aqueous solutions, as well as those collagens which arecommercially available.

Glycosaminoglycans (GAGs) or mucopolysaccharides arehexosamine-containing polysaccharides of animal origin that have atissue specific distribution, and therefore may be used to helpdetermine the tissue specificity of the morphogen-stimulateddifferentiating cells. Reaction with the GAGs also provides collagenwith another valuable property, i.e., inability to provoke an immunereaction (foreign body reaction) from an animal host.

Chemically, GAGs are made up of residues of hexoamines glycosidicallybound and alternating in a more-or-less regular manner with eitherhexouronic acid or hexose moieties (see, e.g., Dodgson et al. inCarbohydrate Metabolism and its Disorders (Dickens et al., eds.) Vol. 1,Academic Press (1968)). Useful GAGs include hyaluronic acid, heparin,heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatansulfate, and keratin sulfate. Other GAGs also can be used for formingthe matrix described herein, and those skilled in the art will eitherknow or be able to ascertain other suitable GAGs using no more thanroutine experimentation. For a more detailed description ofmucopolysaccharides, see Aspinall Polysaccharides, Pergamon Press,Oxford (1970).

Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

Insolubility of the collagen-GAG products can be raised to the desireddegree by covalently cross-linking these materials, which also serves toraise the resistance to resorption of these materials. In general, anycovalent cross-linking method suitable for cross-linking collagen alsois suitable for cross-linking these composite materials, althoughcrosslinking by a dehydrothermal process is preferred.

Formulation Considerations

The devices of the invention can be formulated using any of the methodsdescribed in the art for formulating ostegenic devices. See, forexample, U.S. Pat. No. 5,266,683, the disclosure of which isincorporated herein by reference. Briefly, osteogenic protein typicallyis dissolved in a suitable solvent and combined with the matrix. Thecomponents are allowed to associate. Typically, the combined materialthen is lyophilized, with the result that the osteogenic protein isdisposed on, or adsorbed to the surfaces of the matrix. Usefulsolubilizing solvents include, without limitation, anethanoltrifluoroacetic acid solution, e.g., 47.5% EtOH/0.01% TFA; andacetonitrile/TFA solution, ethanol or ethanol in water, andphysiologically buffered saline solutions. Formulations in an acidicbuffer can faciliate adsorption of OP1 onto the matrix surface. For thereplacement body part devices of the invention, the currently preferredformulation protocol is incubation of matrix and osteogenic protein inan ethanol/TFA solution (e.g., 30-40% EtOH/0.01-0.1%TFA) for 24 hours,followed by lyophilization. This procedure is sufficient to adsorb orprecipitate 70-90% of the protein onto the matrix surface.

The quantity of osteogenic protein used will depend on the size ofreplacement device to be used and on the specific activity of theosteogenic protein. Typically, 0.5 mg-100 mg/10 g of matrix, dry weight,can be used to advantage.

In addition to osteogenic proteins, various growth factors, hormones,enzymes, therapeutic compositions, antibiotics, or other bioactiveagents also can be adsorbed onto, or impregnated within, a substrate andreleased over time when implanted and the matrix slowly is absorbed.Thus, various known growth factors such as EGF, PDGF, IGF, FGF, TGF-a,and TGF-b can be released in vivo. The matrix can also be used torelease chemotherapeutic agents, insulin, enzymes, enzyme inhibitors orchemotactic-chemoattractant factors.

Protein Considerations

As defined herein, the osteogenic proteins useful in the composition andmethods of the invention include the family of dimeric proteins havingendochondral bone activity when implanted in a mammal in associationwith a matrix and which comprise a subclass of the "super family" of"TGFβ-like" proteins. The natural-sourced osteogenic protein in itsmature, native form is a glycosylated dimer typically having an apparentmolecular weight of about 30-36 kDa as determined by SDS-PAGE. Whenreduced, the 30 kDa protein gives rise to two glycosylated peptidesubunits having apparent molecular weights of about 16 kDa and 18 kDa.In the reduced state, the protein has no detectable osteogenic activity.The unglycosylated protein, which also has osteogenic activity, has anapparent molecular weight of about 27 kDa. When reduced, the 27 kDaprotein gives rise to two unglycosylated polypeptides having molecularweights of about 14 kDa to 16 kDa capable of inducing endochondral boneformation in a mammal. Useful sequences include those comprising theC-terminal 102 amino acid sequences of DPP (from Drosophila), Vg1 (fromXenopus), Vgr-1 (from mouse), the OP1 and OP2 proteins, proteins (seeU.S. Pat No. 5,011,691 and Oppermann et al., as well as the proteinsreferred to as BMP2, BMP3, BMP4 (see WO88/00205, U.S. Pat. No. 5,013,649and WO91/18098), BMP5 and BMP6 (see WO90/11366, PCT/US90/01630 and BMP8and 9.

The members of this family of proteins share a conserved six or sevencysteine skeleton in the C-terminal region. See, for example, 335-431 ofSeq. ID No. 1 and whose sequence defines the six cysteine skeletonresidues referred to herein as "OPS", or residues 330-431 of Seq. ID No.1, comprising 102 amino acids and whose sequence defines the sevencysteine skeleton.

This family of proteins includes longer forms of a given protein, aswell as phylogenetic, e.g., species and allelic variants andbiosynthetic mutants, including addition and deletion mutants andvariants, such as those which may alter the conserved C-terminalcysteine skeleton, provided that the alteration still allows the proteinto form a dimeric species having a conformation capable of inducing boneformation in a mammal when implanted in the mammal in association with amatrix. In addition, the osteogenic proteins useful in devices of thisinvention may include forms having varying glycosylation patterns andvarying N-termini, may be naturally occurring or biosyntheticallyderived, and may be produced by expression of recombinant DNA inprocaryotic or eucaryotic host cells. The proteins are active as asingle species (e.g., as homodimers), or combined as a mixed species,including heterodimers.

In one embodiment, the osteogenic protein contemplated herein comprisesOP1 or an OP1-related sequence. Useful OP1 sequences are recited in U.S.Pat. Nos. 5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et al. (1990)EMBO J 9: 2085-2093; and Sampath et al. (1993) PNAS 90: 6004-6008. OP-1related sequences include xenogenic homologs, e.g.; 60A, fromDrosophila, Wharton et al. (1991) PNAS 88: 9214-9218; and proteinssharing greater than 60% identity with OP1 in the C-terminal sevencysteine domain, preferably at least 65% identity. Examples of OP-1related sequences include BMP5, BMP6 (and its species homolog Vgr-1,Lyons et al. (1989) PNAS 86: 4554-4558), Celeste, et al. (1990) PNAS 87:9843-9847 and PCT international application WO93/00432; OP-2 (Ozkaynaket al. (1992) J.Biol. Chem. 267: 13198-13205) As will be appreciated bythose having ordinary skill in the art, chimeric constructs readily canbe created using standard molecular biology and mutagenesis techniquescombining various portions of different morphogenic protein sequences tocreate a novel sequence, and these forms of the protein also arecontemplated herein.

In another preferred aspect, the invention contemplates osteogenicproteins comprising species of polypeptide chains having the genericamino acid sequence herein referred to as "OPX" which accommodates thehomologies between the various identified species of the osteogenic OP1and OP2 proteins, and which is described by the amino acid sequencepresented below and in Sequence ID No 3.

    ______________________________________                                        Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe                                        1        5           10                                                      Xaa Asp Leu Gly Trp Xaa Asp Trp Xaa Ile                                                15           20                                                      Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys                                                25           30                                                      Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser                                                35           40                                                      Xaa Met Asn Ala Thr Asn His Ala Ile Xaa                                                45           50                                                      Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa                                                55           60                                                      Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr                                                65           70                                                      Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa                                                75           80                                                      Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys                                                85           90                                                      Xaa Arg Asn Met Val Val Xaa Ala Cys Gly                                                95          100                                                          Cys His,                                                                  ______________________________________                                    

and wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaaat res. 11=(Mg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In still another preferred aspect, one or both of the polypeptide chainsubunits of the osteogenerically active dimer is encoded by nucleicacids which hybridize to DNA or RNA sequences encoding the active regionof OP1 under stringent hybridization conditions. As used herein,stringent hybridization conditions are defined as hybridization in 40%formamide, 5× SSPE, 5× Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1× SSPE, 0.1% SDS at 50° C.

Given the foregoing amino acid and DNA sequence information, the levelof skill in the art, and the disclosures of numerous publications onosteogenic proteins, including U.S. Pat. No. 5,011,691 and published PCTspecification US 89/01,469, published Oct. 19, 1989, various DNAs can beconstructed which encode at least the active domain of an osteogenicprotein useful in the devices of this invention, and various analogsthereof (including species and allelic variants and those containinggenetically engineered mutations), as well as fusion proteins, truncatedforms of the mature proteins, deletion and addition mutants, and similarconstructs which can be used in the devices and methods of theinvention. Moreover, DNA hybridization probes can be constructed fromfragments of any of these proteins, or designed de novo from the genericsequence. These probes then can be used to screen different genomic andcDNA libraries to identify additional osteogenic proteins useful in theprosthetic devices of this invention.

The DNAs can be produced by those skilled in the art using well knownDNA manipulation techniques involving genomic and cDNA isolation,construction of synthetic DNA from synthesized oligonucleotides, andcassette mutagenesis techniques. 15-100 mer oligonucleotides may besynthesized on a DNA synthesizer, and purified by polyacrylamide gelelectrophoresis (PAGE) in Tris-Borate-EDTA buffer. The DNA then may beelectroeluted from the gel. Overlapping oligomers may be phosphorylatedby T4 polynucleotide kinase and ligated into larger blocks which mayalso be purified by PAGE.

The DNA from appropriately identified clones then can be isolated,subcloned (preferably into an expression vector), and sequenced.Plasmids containing sequences of interest then can be transfected intoan appropriate host cell for protein expression and furthercharacterization. The host may be a procaryotic or eucaryotic cell sincethe former's inability to glycosylate protein will not destroy theprotein's morphogenic activity. Useful host cells include E. coli,Saccharomyces, the insect/baculovirus cell system, myeloma cells, CHOcells and various other mammalian cells. The vectors additionally mayencode various sequences to promote correct expression of therecombinant protein, including transcription promoter and terminationsequences, enhancer sequences, preferred ribosome binding sitesequences, preferred mRNA leader sequences, preferred signal sequencesfor protein secretion, and the like.

The DNA sequence encoding the gene of interest also may be manipulatedto remove potentially inhibiting sequences or to minimize unwantedsecondary structure formation. The recombinant osteogenic protein alsomay be expressed as a fusion protein. After being translated, theprotein may be purified from the cells themselves or recovered from theculture medium. All biologically active protein forms comprise dimericspecies joined by disulfide bonds or otherwise associated, produced byfolding and oxidizing one or more of the various recombinant polypeptidechains within an appropriate eucaryotic cell or in vitro afterexpression of individual subunits. A detailed description of osteogenicproteins expressed from recombinant DNA in E. coli and in numerousdifferent mammalian cells is disclosed in U.S. Pat. No. 5,266,963, thedisclosure of which is hereby incorporated by reference.

Alternatively, osteogenic polypeptide chains can be synthesizedchemically using conventional peptide synthesis techniques well known tothose having ordinary skill in the art. For example, the proteins may besynthesized intact or in parts on a solid phase peptide synthesizer,using standard operating procedures. Completed chains then aredeprotected and purified by HPLC (high pressure liquid chromatography).If the protein is synthesized in parts, the parts may be peptide bondedusing standard methodologies to form the intact protein. In general, themanner in which the osteogenic proteins are made can be conventional anddoes not form a part of this invention.

Exemplification

The means for making and using the matrices and devices of theinvention, as well as other material aspects concerning the nature andutility of these compositions, including how to make and how to use thesubject matter claimed, will be further understood from the following,which constitutes the best mode currently contemplated for practicingthe invention. It will be appreciated that the invention is not limitedto such exemplary work or to the specific details set forth in theseexamples.

In the exemplification, a hemi-joint reconstruction of an articulatingsynovial joint is resected into an existing joint locus. As will beappreciated by those having ordinary skill in the art, the methods andcompositions of the invention equally can be applied to the formation ofreplacement body parts other than skeletal joints, as well as toskeletal joints other than articulating or synovial joints. Moreover, ifdesired, a replacement autogenous joint can be constructed in therecipient first by placing the device of the invention at anotherconvenient locus distal to the defect site, for a time sufficient toinduce formation of the replacement body part, and the autogenous bodypart thus formed then sutured into the joint locus for use.

EXAMPLE 1 Reconstruction of a Mammalian Hemi-Joint

New Zealand white rabbits were used as the experimental model. Standardorthopedic surgical equipment and procedures were used.

As depicted in FIG. 2A, joint defects were created in a recipient bysurgically resecting the entire gleno-humeral hemiarticular complex withthe proximal two-thirds of the humerus. Allografts for implantation wereprepared from hemi-joints excised from a donor animal with the articularsurface of the glenohumoral joint. All allografts were extracted inethanol and lyophilized using standard procedures, and as describedherein above, to destroy the pathogenicity and antigenicity of thematerial. Specifically, intact joint complexes were excised, demarraowedand ethanol treated by exposure to 200 ml-500 ml of 200 proof ethanolfor 72 hours at 40 C. Fresh ethanol was provided every 6-8 hours.Following ethanol treatment, the matrix was lyophilized and rehydratedin ethanol/TFA, with or without osteogenic protein. The treatedhemi-joints comprised devitalized bone, articular cartilage, ligament,tendon, synovialcapsule and synovial membrane tissue.

As illustrated in FIG. 2B, all lyophilized, osteogenic protein-treatedallografts were coated with OP-1 as described in U.S. Pat. No.5,011,691. Specifically, mature, dimeric recombinant OP-1 (rhOP1) wassolubilized in an acetonitrile trifluoro-acetic acid solution, combinedwith the lyophilized allograft, and implanted. 15-20 mg protein/8-10 gmatrix, dry weight, was used. The distal bone portions of all allograftswere secured in place with a four hole titanium miniplate. A meticuloussurgical reconstruction of the joint capsule was performed by suturingthe lyophilized capsule ends to the endogenous capsule using standardsurgical procedures well established in the art using standard surgicalprocedures well established in the art. This recreated an intact capsuleand synovial lining, thereby restoring the synovial milieu of thegrafted articular surface. Motion was permitted almost immediately aftersurgery, again to restore normal joint conditions.

In some animals, local muscle flaps (cutaneous maximus muscle; FIG. 2C)were incorporated into the region of the defect by threading muscle intothe marrow cavity of the allograft as depicted in FIG. 2D, using themethod of Khouri as described in U.S. Pat. No. 5,067,963 the disclosureof which is incorporated herein by reference. Briefly, vascularized andconvenient muscle flaps were dissected using standard procedures wellknown to the practitioner in reconstructive surgery, so as to maintain aperfusing blood supply, and threaded inside the bone marrow cavities ofthe allografts.

Preliminary evaluations of the reconstructed hemi-joints were obtainedby serial weekly radiographs using X-ray, and/or magnetic resonanceimaging (MRI). Histological and mechanical confirmatory evaluations wereconducted upon sacrifice at 5 weeks and 6 months after surgery.

Mechanical evaluations involved standard range of motion (ROM)measurements obtained serially until sacrifice. Histological evaluationsinvolved staining sagital sections through the harvested allograftsusing standard techniques.

Briefly, identification of bona fide articular cartilage can beaccomplished using ultrastructural and/or biochemical parameters. Forexample, articular cartilage forms a continuous layer of cartilagetissue possessing identifiable zones. The superficial zone ischaracterized by chondrocytes having a flattened morphology and anextracellular network which does not stain, or stains poorly, withtoluidine blue, indicating the relative absence of sulphatedproteoglycans. Chondrocytes in the mid and deep zones have a sphericalappearance and the matrix contains abundant sulphated proteoglycans, asevidenced by staining with toluidine blue. Collagen fibers are presentdiffusely throughout the matrix. The chondrocytes possess abundant roughendoplasmic reticulum and are surrounded by extracellular network. Thepericellular network contains numerous thin non-banded collagen fibers.The collagen in the interterritorial network is less compacted andembedded in electron translucent amorphous material, similar toarticular cartilage. Collagen fibers in the interterritorial region ofthe network exhibit the periodic banding characteristic of collagenfibers in the interterritorial zone of cartilage tissue.

Biochemically, the presence of Type II and Type IX collagen in thecartilage tissue is indicative of the differentiated phenotype ofchondrocytes. The presence of Type II and/or Type IX collagen can bedetermined by standard gel electrophoresis, Western blot analysis and/orimmunohisto-chemical staining using, for example, commercially availableantibody. Other biochemical markers include hematoxylin, eosin,Goldner's Trichrome and Safranin-O.

Articular cartilage regeneration was evaluated histologically in theexamples described herein using glycosaminoglycan-specific stains andtechniques well-known in the art or the initial histologic evaluation,the defect sites were bisected lengthwise through the center of thedefect. The resulting halves and surrounding tissue were embedded inparaffin and sectioned across the center of the defect. One half of eachdefect was utilized for histological staining with toluidine blue and/orhematoxlin and eosin, Goldner's Trichrome and Safranin-O. The other halfwas used in preparing sections for immunostaining. Histologicalevaluations involved assessment of: glycosaminoglycan content in therepair cartilage; cartilage and chondrocyte morphology; and, structuralintegrity and morphology at the defect interface. The morphology of therepair cartilage was exhibited for the type of cartilage formed:articular vs. fibrotic by evaluating glycosaminoglycan content, degreeof cartilage deposition, and the like.

Histological evaluations using standard methodologies well characterizedin the art also allows assessment of new bone and bone marrow formation.See, for example, U.S. Pat. No. 5,266,683, the disclosure of which isincorporated hereinabove by reference. Similarly, ligament and synovialcapsule integrity can be monitored by MRI, as well as by histology uponsacrifice.

EXAMPLE 2 Five Weeks Duration (Short Term)

For the 5 week study, four groups with 10 rabbits per group wereimplanted with lyophilized allografts. See FIGS. 3A, 3B, 3C, and 3D. InGroup 1, control lyophilized allograft 30 free of osteogenic protein,was implanted (FIG. 3A). In Group 2, experimental lyophilized allograft31 was impregnated with OP-1 prior to implantation (FIG. 3B). In Group3, control lyophilized allograft 30 free of osteogenic protein, wasimplanted, with muscle flap 32 threaded into marrow cavity 33 (FIG. 3C).In Group 4, experimental lyophilized allograft 31 was impregnated withOP-1 prior to implantation, and muscle flap 32 was threaded within themarrow cavity 33 (FIG. 3D).

As stated above, graft healing was followed non-invasively with serialX-rays and standard MRI (magnetic resonance imaging). By X-rayassessment, allografts treated with osteongic protein had a noticeablythickened cortex by 1 week post-operative, as compared with controlallografts (Groups 1, 3) which evidenced only a thin egg-shell-likecortex. By four weeks the majority control allografts had fractured andwere unstable. In contrast, OP-1 treated allografts (Groups 2, 4)remained stable.

MRI also was used as a non-invasive means for following reformation ofarticular cartilage in the allografts. A dark signal produced by MRIrepresents absent or nonviable cartilage, while a bright signalindicates live, viable cartilage. Control allografts produced only adark signal, when tested at 1, 3 and 5 weeks post-operative. These MRIfindings were confirmed by histological analysis performed at 5 weekspost-operative. Sagital sectioning through control allografts showed adegenerated articular surface with no live cells.

By contrast, the MRI findings of the articular caps from OP-1-treatedallografts showed a bright signal by week 3 post-operative, indicatingregeneration of viable articular cartilage. Histological analysis of theOP-1-treated allografts at week 5 revealed a layer of newly generatedarticular cartilage on top of the allograft matrix. The allografts ofGroup 4 showed somewhat thicker cartilage layers than those of Group 2,suggesting that the addition of the muscle flap may further enhance therate of joint regeneration.

Additionally, joints regenerated with the OP-1-treated allograftsregained near normal range of motion by the time they were harvested at5 weeks post-reconstruction. The near normal range of motion also isindicative of the presence of lubricating synovial fluid. By contrast,the harvested control allografts were stiff and contracted at harvest.Thus, hemi-joint replacement devices of the invention succeeded informing mechanically and functionally viable replacement joints, with anintact capsule, and synovium, and functioning ligament, bone andarticular cartilage tissue. In the absence of osteogenic protein, theallografts, while not rejected by the donor, are insufficient on theirown to generate a functional, weight bearing joint

EXAMPLE 3 Six Months Duration--(Long Term)

For the 6 month study, the variable of shaving off the old cartilaginouscap in the lyophilized allografts was introduced. Briefly, this wasaccomplished by mechanically sharing the articular cartilage cap of thejoint surface.

The following groups were used with 4 rabbits per group: in Group 5,lyophilized allograft 34 with shaved articular surface, and muscle flap32 were implanted (see FIG. 4A); in Group 6, control lyophilizedallograft 30 with non-shaved articular surface, and muscle flap 32 wereimplanted (see FIG. 4B); in Group 7, lyophilized allograft 35 withshaved articular surface and OP-1, and muscle flap 36 treated with OP-1were implanted (see FIG. 4C); and, in Group 8, lyophilized allograft 37with a non-shaved articular surface and OP-1, and muscle flap 36 treatedwith OP-1 were implanted (see FIG. 4D). Grafts in Groups 5-8 wereharvested at 6 months after surgery.

Based upon pre-harvest imaging studies, the results collected by 3months post-operative are consistent with the above-described resultscollected at 5 weeks. Intact allografts treated with OP-1 (Group 8)regenerated a live cartilaginous articular surface by 3 weeks whenevaluated using MRI. This articular cap is still present and even betterdeveloped at 3 months. Without OP-1 treatment of the allograft, (Group6) there was negligible cartilage regeneration relative to the OP-1treated groups.

Similarly, Group 8 rabbits (allograft+OP1, non-shaved) regained nearnormal range of motion (greater than 80%) in the reconstructed joint.Group 7 rabbits (allograft+OP1, shaved) achieved only 50% range ofmotion, and Groups 5 and 6 (no OP1) achieved less than 30%.

As determined by histology, the devices of the invention were competentto induce and maintain both bone and articular cartilage formation inthe appropriate context to one another in a long term study (greaterthan 6 months). Specifically, the rabbits of Group 8, demonstratedarticular cartilage formation on the surface of bone, as evidencedmorphologically by the presence of resting, central and deeper zonechondrocytes. By contrast, in groups treated only with muscle flap,(Group 5 and 6) muscle was replaced with scar tissue. In the groupstreated with shaved bone matrices, no significant cartilage regenerationwas identified, demonstrating the requirement for cartilage-specificresidues in articular cartilage formation in a non-vascularized mileiu.

In both the short term and long term study, mechanically andfunctionally viable synovial joints resulted from the reconstructedhemijoints treated with osteogenic protein, as evidenced by morphologyand biochemistry. In addition, new tissue formed, including articularcartilage, corresponding in shape, kind and structural relationship tothe residues in the devitalized tissue which formed the matrix of thedevice. Collectively, these examples demonstrate that a devicecomprising osteogenic protein and an off-the-shelf, non-viablelyophilized, devitalized matrix can be transformed into aviable,mechanically and structurally functional replacement body part structurecomprising plural distinct newly formed tissues which assume the shapeand function of the original tissue. The device can restore normalfunction to a destroyed body part, including a destroyed skeletal joint,restoring mechanically and functionally viable plural distinct tissues,including bone and bone marrow, articular cartilage, ligament, tendon,synovial capsule and synovial membrane tissue. Moreover, these tissuesare restored under substantially physiological conditionsincluding, forexample, from responding cells present in a synovial environment, andwithout exposure to avascularized muscle flap.

A device comprising osteogenic protein-treated matrices, includinglyophilized allografts or xenografts as disclosed herein can lead to theformation of a new, mechanically, structurally and functionally viablereplacement tissue, and to replacement body parts comprising pluraldistinct tissues, populated by the host cells, and without any of thelimitations of prosthetic materials.

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 3                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1822 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: HOMO SAPI - #ENS                                                (F) TISSUE TYPE: HIPPOC - #AMPUS                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 49..1341                                                        (C) IDENTIFICATION METHOD: - # experimental                         #/function= "OSTEOGENIC PROTEIN"                                                             /product=- # "OP1"                                                            /evidence=- # EXPERIMENTAL                                                    /standard.sub.-- - #name= "OP1"                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #CAC GTG      57AGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG                    #                 Met - # His Val                                             # 1                                                                           - CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TT - #C GTG GCG CTC TGG GCA          105                                                                          Arg Ser Leu Arg Ala Ala Ala Pro His Ser Ph - #e Val Ala Leu Trp Ala           #      15                                                                     - CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GA - #C TTC AGC CTG GAC AAC          153                                                                          Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala As - #p Phe Ser Leu Asp Asn           # 35                                                                          - GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CT - #C CGC AGC CAG GAG CGG          201                                                                          Glu Val His Ser Ser Phe Ile His Arg Arg Le - #u Arg Ser Gln Glu Arg           #                 50                                                          - CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TT - #G GGC TTG CCC CAC CGC          249                                                                          Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Le - #u Gly Leu Pro His Arg           #             65                                                              - CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TC - #G GCA CCC ATG TTC ATG          297                                                                          Pro Arg Pro His Leu Gln Gly Lys His Asn Se - #r Ala Pro Met Phe Met           #         80                                                                  - CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GA - #G GGC GGC GGG CCC GGC          345                                                                          Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Gl - #u Gly Gly Gly Pro Gly           #     95                                                                      - GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GT - #C TTC AGT ACC CAG GGC          393                                                                          Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Va - #l Phe Ser Thr Gln Gly           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TT - #C CTC ACC GAC GCC GAC          441                                                                          Pro Pro Leu Ala Ser Leu Gln Asp Ser His Ph - #e Leu Thr Asp Ala Asp           #               130                                                           - ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CA - #T GAC AAG GAA TTC TTC          489                                                                          Met Val Met Ser Phe Val Asn Leu Val Glu Hi - #s Asp Lys Glu Phe Phe           #           145                                                               - CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TT - #T GAT CTT TCC AAG ATC          537                                                                          His Pro Arg Tyr His His Arg Glu Phe Arg Ph - #e Asp Leu Ser Lys Ile           #       160                                                                   - CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TT - #C CGG ATC TAC AAG GAC          585                                                                          Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Ph - #e Arg Ile Tyr Lys Asp           #   175                                                                       - TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TT - #C CGG ATC AGC GTT TAT          633                                                                          Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Ph - #e Arg Ile Ser Val Tyr           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TC - #G GAT CTC TTC CTG CTC          681                                                                          Gln Val Leu Gln Glu His Leu Gly Arg Glu Se - #r Asp Leu Phe Leu Leu           #               210                                                           - GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GG - #C TGG CTG GTG TTT GAC          729                                                                          Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gl - #y Trp Leu Val Phe Asp           #           225                                                               - ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AA - #T CCG CGG CAC AAC CTG          777                                                                          Ile Thr Ala Thr Ser Asn His Trp Val Val As - #n Pro Arg His Asn Leu           #       240                                                                   - GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GG - #G CAG AGC ATC AAC CCC          825                                                                          Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gl - #y Gln Ser Ile Asn Pro           #   255                                                                       - AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CC - #C CAG AAC AAG CAG CCC          873                                                                          Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pr - #o Gln Asn Lys Gln Pro           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GT - #C CAC TTC CGC AGC ATC          921                                                                          Phe Met Val Ala Phe Phe Lys Ala Thr Glu Va - #l His Phe Arg Ser Ile           #               290                                                           - CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AA - #C CGC TCC AAG ACG CCC          969                                                                          Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln As - #n Arg Ser Lys Thr Pro           #           305                                                               - AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GT - #G GCA GAG AAC AGC AGC         1017                                                                          Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Va - #l Ala Glu Asn Ser Ser           #       320                                                                   - AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GA - #G CTG TAT GTC AGC TTC         1065                                                                          Ser Asp Gln Arg Gln Ala Cys Lys Lys His Gl - #u Leu Tyr Val Ser Phe           #   335                                                                       - CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GC - #G CCT GAA GGC TAC GCC         1113                                                                          Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Al - #a Pro Glu Gly Tyr Ala           340                 3 - #45                 3 - #50                 3 -       #55                                                                           - GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CC - #T CTG AAC TCC TAC ATG         1161                                                                          Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pr - #o Leu Asn Ser Tyr Met           #               370                                                           - AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CT - #G GTC CAC TTC ATC AAC         1209                                                                          Asn Ala Thr Asn His Ala Ile Val Gln Thr Le - #u Val His Phe Ile Asn           #           385                                                               - CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CC - #C ACG CAG CTC AAT GCC         1257                                                                          Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pr - #o Thr Gln Leu Asn Ala           #       400                                                                   - ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AA - #C GTC ATC CTG AAG AAA         1305                                                                          Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser As - #n Val Ile Leu Lys Lys           #   415                                                                       - TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TG - #C CAC TAGCTCCTCC              1351                                                                          Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cy - #s His                           420                 4 - #25                 4 - #30                           - GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CC - #TTGGCCAG       1411                                                                          - GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA AC - #TTTAAAGG       1471                                                                          - TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CA - #GTGGCAGC       1531                                                                          - ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AA - #AAAACAAC       1591                                                                          - GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TG - #CACGGACT       1651                                                                          - CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GG - #AGGAAGGG       1711                                                                          - GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CC - #GGAAGTTC       1771                                                                          #           1822CACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 431 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala         #                 15                                                          - Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser         #             30                                                              - Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser         #         45                                                                  - Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu         #     60                                                                      - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro         # 80                                                                          - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Gly Gly         #                 95                                                          - Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Ty - #r Lys Ala Val Phe Ser         #           110                                                               - Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln As - #p Ser His Phe Leu Thr         #       125                                                                   - Asp Ala Asp Met Val Met Ser Phe Val Asn Le - #u Val Glu His Asp Lys         #   140                                                                       - Glu Phe Phe His Pro Arg Tyr His His Arg Gl - #u Phe Arg Phe Asp Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Al - #a Ala Glu Phe Arg Ile         #               175                                                           - Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp As - #n Glu Thr Phe Arg Ile         #           190                                                               - Ser Val Tyr Gln Val Leu Gln Glu His Leu Gl - #y Arg Glu Ser Asp Leu         #       205                                                                   - Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Se - #r Glu Glu Gly Trp Leu         #   220                                                                       - Val Phe Asp Ile Thr Ala Thr Ser Asn His Tr - #p Val Val Asn Pro Arg         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - His Asn Leu Gly Leu Gln Leu Ser Val Glu Th - #r Leu Asp Gly Gln Ser         #               255                                                           - Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Ar - #g His Gly Pro Gln Asn         #           270                                                               - Lys Gln Pro Phe Met Val Ala Phe Phe Lys Al - #a Thr Glu Val His Phe         #       285                                                                   - Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Ar - #g Ser Gln Asn Arg Ser         #   300                                                                       - Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Me - #t Ala Asn Val Ala Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Ly - #s Lys His Glu Leu Tyr         #               335                                                           - Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Tr - #p Ile Ile Ala Pro Glu         #           350                                                               - Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asn         #       365                                                                   - Ser Tyr Met Asn Ala Thr Asn His Ala Ile Va - #l Gln Thr Leu Val His         #   380                                                                       - Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cy - #s Cys Ala Pro Thr Gln         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp As - #p Ser Ser Asn Val Ile         #               415                                                           - Leu Lys Lys Tyr Arg Asn Met Val Val Arg Al - #a Cys Gly Cys His             #           430                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 102 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..102                                                #/label= OPX) OTHER INFORMATION:                                              #"WHEREIN EACH XAA IS INDEPENDENTLY SELECTED                                  #GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS                                                  AS DEFINE - #D IN THE SPECIFICATION"                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xa - #a Asp Leu Gly Trp Xaa         #                15                                                           - Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Al - #a Tyr Tyr Cys Glu Gly         #            30                                                               - Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met As - #n Ala Thr Asn His Ala         #        45                                                                   - Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pr - #o Xaa Xaa Val Pro Lys         #    60                                                                       - Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xa - #a Ser Val Leu Tyr Xaa         #80                                                                           - Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xa - #a Arg Asn Met Val Val         #                95                                                           - Xaa Ala Cys Gly Cys His                                                                 100                                                               __________________________________________________________________________

What is claimed is:
 1. A device for forming a stable, functional,vitalized articulating skeletal joint at a joint defect site in amammal, the device comprising:a biocompatible, bioresorbable,devitalized matrix, excised from a mammalian donor articulating skeletaljoint and comprising, an articulating surface, and, plural distincttissues including at least one non-mineralized tissue, said tissuescapable of essentially maintaining their shape and relationships whenused as a replacement joint, and having dimensions and structuralrelationships to each other which correspond anatomically to thearticulating skeletal joint to be replaced, and, disposed on or withinsaid matrix, substantially pure exogenous osteogenic protein in anamount sufficient to induce formation of a new vitalized articulatingsurface and new vitalized plural distinct tissues thereby to permitregeneration of said stable, functional, vitalized articulating skeletaljoint at said defect site.
 2. A device for forming plural distinctnon-mineralized tissues in a stable, functional, vitalized articulatingskeletal joint in vivo, the device comprising:a biocompatible,bioresorbable, devitalized matrix excised from a mammalian donorarticulating skeletal joint and comprising, an articulating surface,and, plural distinct non-mineralized tissues capable of essentiallymaintaining their shape and relationships when used as a replacementjoint, said tissues having dimensions and structural relationships toeach other which correspond anatomically to the articulating skeletaljoint to be replaced, and, disposed on or within said matrix,substantially pure exogenous osteogenic protein in an amount sufficientto induce formation of a new vitalized articulating surface and newvitalized plural distinct non-mineralized tissues thereby to permitregeneration of said stable, functional, vitalized articulating skeletaljoint at said defect site.
 3. The device of claim 1 wherein said pluraldistinct tissues comprise articular cartilage and bone.
 4. The device ofclaim 1 or 2 wherein said non-mineralized skeletal joint tissue isselected from the group consisting of articular cartilage, ligament,tendon, intervertebral discs, joint capsule and synovial membranetissue.
 5. The device of claim 1 or 2 wherein said skeletal jointcomprises a synovial joint.
 6. The device of claim 1 or 2 wherein saidnon-mineralized tissue comprises an avascular tissue.
 7. The device ofclaim 1 or 2 wherein said matrix comprises devitalized allogenic orxenogenic tissue.
 8. The device of claim 1 or 2 wherein said devicefurther comprises a matrix selected from the group consisting of:collagen; polymers comprising monomers of lactic acid, glycolic acid,butyric acid or derivatives thereof; and ceramics, hydroxyapatite,tricalcium phosphate, or mixtures thereof.
 9. The device of claim 1 or 2further comprising a material suitable for binding particulate matter toform a moldable solid.
 10. The device of claim 1 or 2 wherein saidexogenous osteogenic protein comprises homodimers or heterodimers ofOP-1, OP-2, BMP2, BMP3, BMP4, BMP5, BMP6, OPX, or naturally sourced andrecombinant derivatives of the foregoing.
 11. A method for restoring astable, functional, vitalized articulating skeletal joint at a jointdefect site in mammal, the method comprising the step of:providing tothe joint defect site, a device comprising a biocompatible,bioresorbable devitalized matrix, excised from a mammalian donorarticulating skeletal joint and comprising, an articulating surface,and, plural distinct tissues including at least one non-mineralizedtissue, said tissues capable of essentially maintaining their shape andrelationships when used as a replacement joint, and having dimensionsand structural relationships to each other which correspond anatomicallyto the articulating skeletal joint to be replaced, and disposed on orwithin said matrix, substantially pure exogenous osteogenic protein inan amount sufficient to induce formation of a new vitalized articulatingsurface and new vitalized plural distinct tissues thereby to permitregeneration of said stable, functional, vitalized articulating skeletaljoint at said defect site.
 12. A method for restoring in a mammal,plural distinct non-mineralized tissues in a skeletal joint, the methodcomprising the step of:providing to the joint defect site, a devicecomprising biocompatible, bioresorbable, devitalized matrix, excisedfrom a mammalian donor articulating skeletal joint and comprising, anarticulating surface, and, plural distinct non-mineralized tissuescapable of essentially maintaining their shape and relationships whenused as a replacement joint, said tissues having dimensions andstructural relationships to each other which correspond anatomically tothe articulating skeletal joint to be replaced, and disposed on orwithin said matrix, substantially pure exogenous osteogenic protein inan amount sufficient to induce formation of a new articulating surfaceand new plural distinct non-mineralized tissues thereby to permitregeneration of said stable, functional, vitalized articulating skeletaljoint at said defect site.
 13. The method of claim 11 wherein saidplural distinct tissues comprise articular cartilage and bone.
 14. Themethod of claim 11 or 12 wherein said matrix comprises devitalizedallogenic or xenogenic tissue.
 15. The method of claim 11 or 12 whereinsaid skeletal joint is a synovial joint.
 16. The method of claim 11 or12 wherein said non-mineralized tissue to be restored comprisesavascular tissue.
 17. The method of claim 11 or 12 wherein saidnon-mineralized tissue to be restored is selected from the groupconsisting of articular cartilage, tendon, ligament, intervertebraldiscs, joint capsule and synovial membrane tissue.
 18. The method ofclaim 11 or 12 wherein said device further comprises a matrix selectedfrom the group consisting of: collagen; polymers comprising lactic acid,butyric acid, glycolic acid or combinations thereof; and ceramics,tricalcium phosphate, hydroxyapatite or mixtures thereof.
 19. The methodof claim 11 or 12 wherein said exogenous osteogenic protein compriseshomodimers or heterodimers of OP-1, OP-2, BMP2, BMP3, BMP4, BMP5, BMP6,OPX, or naturally sourced and recombinant derivatives of the foregoing.